Biomedical subjects

J Cerbón

Publications and source records attributed to J Cerbón.

At least 19 recordsLinked to original sources

PubMed2000

Purification and properties of an acid phosphatase from Entamoeba histolytica HM-1:IMSS.

Entamoeba histolytica contains and secretes acid phosphatase, which has been proposed as a virulence factor in some pathogenic microorganisms. In this work, we purified and characterised a membrane-bound acid phosphatase (MAP) from E. histolytica HM-1:IMSS and studied the effect of different chemical compounds on the secreted acid phosphatase and MAP activities. MAP purification was accomplished by detergent solubilisation, and affinity and ion exchange chromatographies. The enzyme showed a pI of 5.5-6.2, an optimum pH of 5.5, and a Km value of 1.14 mM with p-nitrophenyl phosphate.

Acid Phosphatase↗

PubMed1997

Ecto-nucleotide triphosphatase activity in pathogenic and non-pathogenic Entamoeba: protection from the cytotoxic effects of extracellular ATP.

The nucleotide triphosphatase (NTPase) activity of Entamoeba species and of Entamoeba histolytica strains, was compared. In all cases, with the exception of Entamoeba moshkovskii, the enzyme was activated by Ca2+ and not by Mg2+ and preferentially hydrolysed UTP with decreasing activity for ATP and GTP. The NTPase activity was associated with both the intracellular and the plasma membrane sulphonylbenzoyl-adenosine (FSBA) of trophozoites in which the ATP-site was facing externally, as shown by fluoroinhibition of NTPase activity and protection by the substrate added prior to FSBA. The highest surface activity was found in Entamoeba invadens and in the virulent E. histolytica HM1-A clone (HM1-IMSS passaged thrice through hamster liver). Significant lower activity was observed in non-pathogenic Entamoeba spp. The addition of ATP to cultures of pathogenic amoebae resulted in cell growth inhibition and lysis. This deleterious effect of adding ATP to the cultures was reversed by the addition of Ca2+. ATP hydrolysis by the amoeba may alter extracellular ATP-dependent processes in the host, which may be important for the survival of the amoeba in vivo.

Acid Anhydride Hydrolases↗

PubMed1995

Generation, modulation and maintenance of the plasma membrane asymmetric phospholipid composition in yeast cells during growth: their relation to surface potential and membrane protein activity.

During growth a cyclic exposure of anionic phospholipids to the external surface of the plasma membrane was found. The surface charge density (sigma) increased gradually reaching a maximum in the first 5 h of growth and returned gradually to their initial value at the end of the logarithmic phase of growth (10-12 h). Phosphatidylinositol, that determines to a large extent the magnitude of the sigma, increased 83% in the yeast cells during the first 4 h of growth and returned gradually to their initial level at 10-12 h. During the stationary phase (12-24 h), both sigma and the anionic/zwitterionic phospholipid ratio, remained without any significant variation. The high-affinity H-linked glutamate transport system that behaves as a sensor of the changes in the membrane surface potential (phi) increased its activity in the first 5 h and then decreased it, following with great accuracy the sigma variations and remained without changes during the stationary phase of growth. The phosphatidylserine (PS) relative concentration in the cells (9.0%) did not significantly change during the whole growth curve, but their asymmetric distribution varied, contributing to the changes in sigma. PS facing the outer membrane surface increased 2.45-times during the first 5 h of growth and then returned to their original value at the end of the log phase (12 h). Phosphatidylcholine (PC) remained constant during the whole growth curve (50%), while phosphatidylethanolamine (PE) decreased 3-fold in the first 4 h and then increased to its original value at 10 h. Interestingly, PE at the outer membrane surface remained constant (3% of the total phospholipids) during the whole growth curve. During growth yeast cells change their phospholipid composition originating altered patterns of the plasma membrane phospholipid composition and IN-OUT distribution. This dynamic asymmetry is involved in the regulation of the surface potential and membrane protein activity.

Aminacrine↗

PubMed1994

Surface potential regulation of phospholipid composition and in-out translocation in yeast.

In yeast cells the anionic phospholipids, phosphatidylinositol and phosphatidylserine, determine to a large extent the magnitude of the negative surface charge density (sigma) [Cerbón, J. & Calderón, V. (1990) Biochim. Biophys. Acta 1028, 261-267]. We now report further findings. (a) When the yeast phi out was reduced by increasing the concentration of monovalent (C+) or divalent (C2+) cations in the culture medium, the relative amount of anionic phospholipids increased (45-52%). (b) For each such increment, a corresponding increase in the external surface charge density (sigma) was found, due to the translocation from the cytoplasmic side to the exoplasmic side of the plasma membrane. (c) These changes were reversed when the phi out was increased by reducing the concentration of cations in the culture medium. (d) When the phi out was reduced and phosphatidylserine decarboxylation or phosphatidylinositol degradation were inhibited, to measure synthesis of anionic phospholipids, a 1.4 times further increase in the anionic/zwitterionic phospholipid ratio occurred. As a consequence, a similar increase in the external surface charge (sigma) was found. (e) Under all the conditions studied, the percentage of anionic phospholipid at the external surface of the plasma membrane calculated from the sigma values was 2.3-3.0 times less than that in the cells, indicating that the asymmetric composition (more inside) was maintained. A model for the regulation of the anionic phospholipid composition of the yeast membranes is proposed.

Cell Membrane↗

PubMed1992

Interfacial pH modulation of membrane protein function in vivo. Effect of anionic phospholipids.

In yeast cells, the magnitude of the membrane surface potential (phi) is determined to a large extent by the relative amount of anionic phospholipids (Cerbón and Calderón (1990) Biochim. Biophys. Acta 1028, 261-267). When a significant surface potential exists, the pH at the membrane surface (interfacial pH) will be different to that in the bulk suspending medium. We now report that: (1) In cells with higher phi (phosphatidylinositol-rich cells (PI-rich) and phosphatidylserine-rich cells (PS-rich) a 10-times lower proton concentration in the bulk was enough to achieve the maximum transport activity of H(+)-linked transport systems when compared to normal cells. (2) When the phi was reduced by increasing the concentration of cations in the medium, more protons were required to achieve maximum transport, that is, the pH activity curves shifted downwards to a more acidic pH. (3) The magnitude of the downward pH shift was around 2.5-times higher for the more charged membranes. (4) Around 10-times more KCl than MgCl2 was necessary to give an equivalent pH shift, in agreement with their capacity to reduce the phi of artificial bilayers. The interfacial pH calculated from the values of phi indicates that it was 0.4 pH units lower in the anionic phospholipid rich cells as compared to normal cells. The results indicate that membrane surface potential may explain the complex relationship between pH, ionic strength and membrane protein function. Maximum transport activities were found for glutamate at interfacial pH of 4.2-4.8 and were inhibited at interfacial pH = 3.2-3.4, suggesting that surface groups of the carrier proteins with pK values in the region 3.8-4.2 (aspartyl and glutamyl) are involved in binding and/or release of charged substrates.

Arginine↗

PubMed1992

Nigericin forms highly stable complexes with lithium and cesium.

Nigericin is a monocarboxylic polyether molecule described as a mobile K+ ionophore unable to transport Li+ and Cs+ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li+, Cs+, Na+, Rb+, and K+. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericin-alkali metal cation binding isotherms show faster rates for Li+ and Cs+ than for Na+, K+, and Rb+, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li+ and nigericin-Cs+ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na+, K+, and Rb+ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li+ and Cs+ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K+ greater than Rb+ greater than Na+.

Biological Transport↗

PubMed1991

Changes of the compositional asymmetry of phospholipids associated to the increment in the membrane surface potential.

The contribution of phosphatidylinositol (PI) and phosphatidylserine (PS) to the outer negative membrane surface potential was studied in normal, PS-rich and PI-rich yeast cells. Under carefully defined conditions; PS and PE were quantified by using the non-penetrating chemical probe trinitrobenzenesulfonic acid (TNBS) and the PI by degradation with a specific phospholipase C. An asymmetric distribution of phospholipids in the plasma membrane with more PS (80-90%), PI (70-85%) and PE (70-85%) in the inner leaflet was found. When compared to normal cells there were 3-times more PI and 2-times more PS in the outer leaflet of the PI-rich and PS-rich cells. These values are consistent with the two-times increased surface potential in these cells. Interestingly, the contribution of PI was around twice the contribution of PS to the surface potential in the cells studied. When compared to normal cells there was a two-times increased accessibility of PS to TNBS in the PI-rich cells and the accessibility of PI to phospholipase C was also increased two-times in the PS-rich cells, while the proportion of derivatized PE was similar in all cells. Taking into account that the amount of PI is similar in normal cells and PS-rich cells and the amount of PS is similar in PI-rich cells and normal cells, a charge driven transbilayer transport of acidic phospholipids can be proposed.

Cell Membrane↗

PubMed1991

Anionic phospholipids in the control of the membrane surface potential in Escherichia coli: their influence on transport mechanisms.

1. Phosphatidylserine (PS)-rich Escherichia coli cells were utilized to investigate the role of anionic phospholipids on membrane surface potential and their effect upon active transport mechanisms. 2. It was found that: 3. The transport of inorganic phosphate and glutamate, which depends upon cations, was increased (Km decreases) in PS-rich cells as compared to normal cells. 4. The reduction of the negative surface potential by MgCl2 or by the cationic local anesthetic procaine, brought about a decrement in the uptake of both substrates. 5. When the negative surface potential of the PS-rich cells was reduced, the Km returned back to the values found in normal cells. 6. A direct correlation between the ratio anionic/zwitterionic phospholipids, negative surface potential and increment in the initial rate of transport was found.

Anions↗

PubMed1990

Proton-linked transport systems as sensors of changes in the membrane surface potential.

The kinetic properties of proton linked transport systems and their relation to the membrane surface potential were studied in yeast cells. (1) The negative surface potential of cells rich in anionic phospholipids was found to be 2-times higher than that of control cells; in agreement with their 2-fold increase in the anionic/zwitterionic phospholipid ratio (A/Z). (2) At low external concentration of substrates (high-affinity systems), higher uptake activities were observed for the anions, glutamate, aspartate and phosphate; the zwitterion glycine and the cations lysine and arginine, in both phosphatidylserine and phosphatidylinositol rich cells when compared to control cells. (3) On the other hand, at high external concentration of substrates (low-affinity systems), lower uptake activities were observed for glutamate, aspartate, phosphate and glycine in the cells rich in anionic phospholipids. (4) A decrease in Km without significant alteration in Vmax was found in the high-affinity transport systems that can be explained by the increase in proton concentration at the interface caused by the enhancement in negative surface charge of the cells rich in anionic phospholipids. (5) The mechanisms of the high-affinity proton linked transport systems are compatible with a model which is necessarily ordered, protons before anions. The low-affinity transport systems, on the other hand, follow a random order of binding. The transport systems studied behave as sensors of the changes in surface potential. The reduction of the surface potential reversed the transport alterations with the following sequence: monovalent cations less than divalent cations less than cationic local anesthetics.

Biological Transport↗

PubMed1987

Formation of ion-translocating oligomers by nigericin.

At pH 4.0, greater than 10(-7) M nigericin was found capable of conducting net charge transfer across bimolecular lecithin membranes, with a stoichiometry of three uncharged ionophore moieties per cation. At neutral or alkaline pH, nigericin catalyzed the transfer of net charge through dimer forms. In agreement with these results, quantitative analysis of nigericin-potassium complexes formed at pH 4.0 showed a 3:1 ratio, and a 2:1 ratio at neutral or alkaline pH. A 1:1 stoichiometry was observed when the ionophore complex was not transferred from methanol-water to chloroform. Moreover, 1H-NMR spectra of nigericin-cation complexes formed at pH 4.0, displayed clear-cut chemical shift variations different to those observed at neutral or alkaline pH. Thus, it is apparent that acid pH causes a transition from dimeric to trimeric forms of nigericin-cation complexes. The membrane conductance increased up to ten times when negatively charged phosphatidyl glycerol was used, while the conductance decreased in positively charged cetylpyridinium containing membranes at pH 4.0. These results suggest that the nigericin-K+ oligomeric complex is positively charged. In this respect, pKa values around 8.0 were obtained for the nigericin carboxylate group in media of different dielectric constant, indicating that this chemical group is undissociated under these conditions. Moreover, the values for the complex formation constants as well as the delta G values calculated for the dimers and trimers indicated that such ionophore cation oligomeric complexes are thermodynamically stable.

Anti-Bacterial Agents↗

PubMed1987

Lactate and pyruvate increase the incorporation of [3H]proline into collagen [3H]hydroxyproline in liver slices of CCl4 cirrhotic rats.

Lactate and pyruvate enhanced the incorporation of [3H]proline into collagen [3H]hydroxyproline when added to liver slices of CCl4-treated rats. In addition, pyruvate stimulated the accumulation of cAMP, reaching maximum values after 10 minutes of incubation. Similar results were obtained with newborn rat calvariae, a tissue that normally produces large amounts of type I collagen. In normal liver, which produces relatively small amounts of collagen, lactate had no effect on cAMP levels or collagen synthesis. Pyruvate stimulated the accumulation of cAMP, but had no effect on collagen synthesis. These results indicate that different control mechanisms are involved in regulation of collagen biosynthesis in normal as compared with cirrhotic liver; the latter resembling mesenchymal tissues specialized in collagen production such as newborn rat calvariae.

Animals↗

PubMed1986

Phosphoinositides provide a regulatory mechanism of surface charge and active transport.

Yeast cells, when grown in the presence of arsenate, are capable of accumulating phosphoinositides (PI) at the expense of inhibiting their degradation more than their synthesis. PI levels return to normal when the cells are cultured or exposed to media without arsenate. These reversible changes are employed as a tool to test the effect of inositide accumulation and dynamics on several membrane properties. In the PI-rich cells, phosphate and arsenate transport from low external concentrations (high affinity systems), as well as the transport of glycine, which enter the cells accompanied by protons, were increased. The proton ejection energized by glucose is also enhanced in the PI-rich cells that show a more efficient potassium inflow at pH 4.0-4.5. The membrane surface potential of the PI-rich cells was found to be 2-times higher than that of the normal cells, in agreement with the 2-fold increment in the PI. All the above mentioned alterations in membrane properties are reverted when the PI content of the PI-rich cells is reduced to the level of normal cells. The results show the participation of the phosphoinositides in the formation, maintenance and regulation of the membrane surface potential and their possible influence upon transport mechanisms.

Arsenates↗

PubMed1978